Abstract

Many metabolic pathways are critically regulated during development and aging but little is known about the molecular mechanisms underlying this regulation. One key metabolic cascade in eukaryotes is the mevalonate pathway. It catalyzes the synthesis of sterol and nonsterol isoprenoids, such as cholesterol and ubiquinone, as well as other metabolites. In humans, an age-dependent decrease in ubiquinone levels and changes in cholesterol homeostasis suggest that mevalonate pathway activity changes with age. However, our knowledge of the mechanistic basis of these changes remains rudimentary. We have identified a regulatory circuit controlling the sumoylation state of Caenorhabditis elegans HMG-CoA synthase (HMGS-1). This protein is the ortholog of human HMGCS1 enzyme, which mediates the first committed step of the mevalonate pathway. In vivo, HMGS-1 undergoes an age-dependent sumoylation that is balanced by the activity of ULP-4 small ubiquitin-like modifier protease. ULP-4 exhibits an age-regulated expression pattern and a dynamic cytoplasm-to-mitochondria translocation. Thus, spatiotemporal ULP-4 activity controls the HMGS-1 sumoylation state in a mechanism that orchestrates mevalonate pathway activity with the age of the organism. To expand the HMGS-1 regulatory network, we combined proteomic analyses with knockout studies and found that the HMGS-1 level is also governed by the ubiquitin-proteasome pathway. We propose that these conserved molecular circuits have evolved to govern the level of mevalonate pathway flux during aging, a flux whose dysregulation is associated with numerous age-dependent cardiovascular and cancer pathologies.

ULP-4 exhibits a cytosol-to-mitochondria translocation. (A–C) ULP-4 expression initiates at midembryogenesis in muscles. ULP-4 protein is distributed in the cytoplasm. (D) ULP-4 protein cytoplasm-to-mitochondria translocation in hypodermal cells at L1 stage. Pharyngeal cells also start to express ULP-4 that is sorted to the mitochondria. (E) At L3 BWM cells express ULP-4 as a cytoplasmic protein (arrow). (F) At L4, ULP-4 expression initiates at the HSNs concomitant with ULP-4 translocation to the mitochondria of head and tail muscles. (G) Day-1 adult worms exhibit mitochondrial localization of ULP-4 in HSNs and most of the BWM cells. G, Inset shows a muscle anterior to the HSN soma where ULP-4 is still cytoplasmic. (H) In day-10 adult worms, ULP-4 localizes to the mitochondria of all muscle cells. H, Inset shows mitochondrial ULP-4 in the region as in G. (I–J″) Colocalization of ULP-4 with the mitochondrial outer membrane marker TOM-20::mRFP in muscles demonstrates ULP-4 matrix localization. (I) A merged image of a single muscle cell. (J–J″) A single organelle analysis of TOM-20:mRFP distribution (J), ULP-4::GFP localization (J′), and merged distributions (J″). (K) A comprehensive diagram of ULP-4 expression and localization pattern. In all experiments, n ≥ 15 worms. (Scale bars: 10 µm in A–C, 50 µm in D–H, 10 µm in G and H Insets, 10 µm in I, and 2.5 µm in J.)

HMGS-1 is sumoylated in an age-dependent manner in vivo. (A) ULP-4 and HMGS-1 protein fragments interact in a yeast two-hybrid assay. (B–D) In vitro sumoylation reactions. Lanes labeled with −E2 are control reactions in which UBC9 is absent and −S are controls in which substrate is absent. (B) Time course (in minutes) of HMGS-1 sumoylation. (C) Similar to HMGS-1, human HMGCS1 is sumoylated in vitro. (D) The sumoylation pattern of WT and various lysine-to-arginine HMGS-1 mutants constructed based on sumoylation prediction (). 4KR is a HMGS-1 variant whose four predicted sumoylated lysines were mutated to arginines. Arrows mark the sumoylated-HMGS-1 bands, and asterisks label sumoylation-abolished HMGS-1 variants. (E) An assay for HMGS-1 sumoylation detection in vivo. IB, immunoblot. (F) HMGS-1 sumoylation increases with age and temperature until day 10 of adulthood. Total levels of HMGS-1 were determined by HMGS-1::FLAG::GFP detection from the total lysates. (G) ULP-4 knockdown resulted in increased HMGS-1 sumoylation whereas ULP-4 overexpression (ulp-4 OEx) resulted in a mild decrease of HMGS-1 sumoylation.